Neural stem cells (NSC) have a fascinating biology of precise transition of cellular states from dormant to proliferative to differentiating, which most recently begun to be understood at the cellular and molecular levels. Despite successful identification of genes linked to each of those cellular steps, a comprehensive and multi-dimensional picture on how those genes are regulated in real-time throughout the differentiation stages of NSC is sorely lacking. This multi- disciplinary proposal will combine for the first time the use of two powerful methods to directly assess the transcriptional state of key genes involved in NSC regulation in vivo, at a single cell resolution. The first technique, Multiplex in situ hybridization, co-developed by one of the PIs, allows direct visualization of transcriptional activity of several genes with single cell resolution. The second technique, known as G-Trace, will be used to generate cellular clones labeled with fluorescent proteins that distinctively mark cell lineages according to their temporal generation. In Aim 1, the combination of these two methods in the Drosophila larval brain will be used to create profiles of normal wild type expression of several genes involved in NSC regulation simultaneously. Further computational image analysis tools will be developed to segment images and reduce noise. We will test the application of a spectral bar coding system as a proof-of-principle to expand the detection of hundreds of active genes analyzed at a time. In Aim 2, brain tumors will be induced by knocking-out key transcription factors to track global alterations in gene expression within labeled clones. The expectation is that these combined methods will reveal key properties of the differentiation progression of NSC into several specific neuronal lineages, with far-reaching implications to a better manipulation of stem cells in general and in other organisms, including humans.